There is a large volume of literature on the manufacturing of planar microfabricated devices primarily using electroosmotic, electrokinetic, and/or pressure-driven motions of liquid and particles as the means of fluid transport. Microfluidic devices are considered an enabling technology for low cost, high versatility operations, many of which find great utility in the biotechnology and pharmaceutical industries. Jorgenson et al., Journal of Chromatography, vol. 218, 1981, pp. 209–214 and U.S. Pat. No. 4,908,112 teach the use of microfluidic devices made of silicon having a glass cover to perform separation operations using capillary electrophoresis (CE), a common application for such a device. Their application was the electrophoretic separation of double-stranded DNA fragments labeled with intercolating dye for fluorescence detection. It is known that silicon has limitations for this application because the electric field needed to drive the fluid in a capillary electrophoretic separation device often exceeds the dielectric breakdown field of silicon. U.S. Pat. No. 5,126,022 and U.S. Pat. No. 5,858,188 are exemplary of the use of insulating substrates for light or laser induced fluorescence (LIF) detection systems. Polymeric materials, in particular poly methyl methacrylate (PMMA), have the optical clarity in the visible wavelengths required for such LIF detection systems. It has been demonstrated that PMMA can be injection molded to reproduce features of about 100 μm, a dimension usable in the construction of microfluidic channels in these devices. However, injection molding at dimensions smaller than 100 μm has not been disclosed. These smaller dimensions are especially desirable for capillary electrophoretic separations. These smaller dimensions minimize the spatial spread of the sample plug in sample inlet channels. U.S. Pat. No. 5,885,470, and subsequent patents with the same assignees, disclose polymer devices made of polydimethylsiloxane (PDMS), PMMA, polyurethane, polysulfone and polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC) and polycarbonate. Each of these polymers suffers disadvantages. All these materials are hydrophobic. PDMS and polyurethane are not injection moldable, PVC is typically chemically impure, PMMA and polycarbonate are not UV-transparent and PTFE is typically not optically clear.
The proceedings of the Micro Total Analysis Systems-2000 Symposium, A. Van Den Berg and W. Olthuis, ed., Kluwer Academic Publishers, Dortrecht, 2000, exemplify the progress made in this field, in device fabrication methods, complexity and applications. Microfluidic devices made of glass are exemplified by Zhonghui H. Fan and D. Jed Harrison, Analytical Chemistry, vol. 66, 1994, pp. 177–184 and A. M. Woolley et al., Analytical Chemistry, vol. 69, 1997, pp. 2181–2186. Microfluidic devices made of polymeric substrates are exemplified by R. M. McCormick et al., Analytical Chemistry, vol. 69, 1997, pp. 2626–2630, Hou-pu Chou et al., Solid-State Sensor and Actuator Workshop Proceedings, June 1998, pp. 11–14, C. S. Effenhauser et al. Analytical Chemistry, vol. 69 (1997) 3451–3457, T. D. Boone et al., Solid-State Sensor and Actuator Workshop Proceedings, June 1998, pp. 87–92, and M. A. Roberts et al., Analytical Chemistry, vol. 69, 1997, pp. 2035–2042.
A large fraction of the applications using these devices have been in DNA analysis with fluorescence detection. E. C. Lagally et al., Micro Total Analysis Systems-2000 Symposium, pp. 217–220, discusses high throughput DNA analysis with integrated PCR. C. S. Effenhauser, ibid., discusses DNA restriction enzymes. A. Eckersten el al., Micro Total Analysis Systems-2000 Symposium, pp. 521–524, discusses high throughput single nucleotide polymorphism (SNP) scoring in a disposable microfabricated capillary electrophoresis (CE) device. Other applications include chemiluminescence, electroconductivity and mass spectrometry detection. U.S. Pat. No. 5,637,469 (Wilding et al) teaches sperm sample analysis with chemiluminescence detection. G. Weber et al., Micro Total Analysis Systems-2000 Symposium, pp. 383–386, discusses organic acid separation and detection using electroconductivity detection. T. Miliotis, Micro Total Analysis Systems-2000 Symposium, pp. 387–390, discusses protein and peptide separation using mass spectrometry detection. Commercial systems for microfluidic applications to DNA analysis have also started to appear on the market.
Fluorescence detection, or laser-induced fluorescence (LIF), is a very sensitive detection technique, but it requires that analytes be derivatized with fluorescent dyes. The method has wide applications in DNA analysis in which dye derivatization is standard procedure. On the other hand, UV spectrophotometry as a detection technique is attractive because many analytes of interest are UV chromophores and therefore do not require sample preparation procedures such as tagging analytes with fluorophores. Moreover, UV spectroscopy, being an optical technique, is more robust than techniques such as electroconductivity.
Electroconductivity detection is susceptible to contamination problems on the electrode and is difficult to operate in capillary electrophoresis because of the presence of the high electrophoretic voltage in the background. For this reason, UV detection has a far wider range of applications including the analyses of proteins, small molecules, chiral compounds, etc. The most common substrate material for microstructure fabrication has been Pyrex®, which is not particularly transparent at wavelengths shorter than four hundred nanometers (400 nm). The use of Pyrex® in microfluidic devices has hindered the application of these devices to UV spectroscopy. Likewise, the most popular polymer resins, such as PMMA, used in visible wavelength optical applications are not particularly transparent below three hundred nanometers (300 nm). The transparent polymers in the previously mentioned U.S. Pat. No. 5,885,470 are not UV-transparent except PDMS, which is not injection moldable.
The other hindrance for incorporating UV spectrophotometry as the detection technique is that UV sensitivity is several orders of magnitude lower than that of light fluorescence spectroscopy. Quartz, a material that has been used in conventional chromatography and capillary electrophoresis, requires very high temperatures to bond a cover to a surface having microfabricated structures. There have been some recent efforts to circumvent this problem. T. Nishimoto et al., Micro Total Analysis Systems-2000 Symposium, pp. 395–398, describes a special HF bonding method that allows easier bonding, and the incorporation of a silicon slit in the detection window to cut down background stray light to increase signal to noise intensity. R. J. Jackman, Micro Total Analysis Systems-2000 Symposium, pp. 155–158, has adopted another strategy, the use a layer of photoresist as bonding agent. Still another strategy, disclosed by H. Bjorkman et al., Micro Total Analysis Systems-2000 Symposium, pp. 187–190, uses artificial diamond as the optical window material. Artificial diamond, however, is made by an expensive process.
The scientific literature has been somewhat vague concerning specific methods to seal channels in polymer devices. A common way to seal microfluidic channels in polymer substrates is to simply cover the channels with an adhesive covered tape having low background fluorescence at visible wavelength. The tape is typically bonded to the substrate using UV bonding or similar techniques known in the art. The tape is not an optimal solution for sealing devices because it has limited mechanical strength, and is generally not UV-transparent.
Although prior art polymeric materials suffer certain deficiencies, they are becoming an increasingly popular choice for fabricating microfluidic devices. Prior art polymeric materials are not sufficiently transparent in the ultraviolet. Polymeric materials suffer from the deficiency of lower thermal conductivity than silicon and glass, the original materials of construction for microfluidic devices. A common need for all analytical devices, particularly devices employing ultraviolet detection, is increased sensitivity. The present invention overcomes these deficiencies.